Cell migration is a fundamental biological process and much is now known about mechanisms involved in single cell motility on planar substrates of extracellular matrix. Less well understood is the migration of contiguous groups of cells (e.g., sheets, clusters, and cord-like structures) where cell-cell interactions are involved in the regulation of cell polarity and protrusive activities within the migratory array. The central hypothesis to be tested during the next progress period is that a cadherin complex with links to the intermediate filament cytoskeleton functions as a putative mechanosensory apparatus to direct and orient collective cell movements in the mesendoderm of Xenopus embryos. Progress during the previous project period established the importance of cadherin-dependent cell-cell contact in directing mesendoderm cell polarity, protrusive behaviors and migration on fibronectin (FN) in vivo. Moreover, directed cell movements on FN require force application through cadherins and an intact intermediate filament cytoskeleton. The coordinate involvement of both cadherins and integrins in this process is a key target of this proposal, which seeks to establish the mechanical/cytoskeletal and signaling linkages involved. The proposal has two Specific Aims. First, the relationship between cadherin adhesion, force application and the intermediate filament and actin cytoskeletons will be established in order to identify mechanisms of regulation of cell polarity and directed cell migration. Traction force microscopy (TFM) and paramagnetic bead-pull experiments will be used to define cell-cell adhesive forces required to maintain directional migration on FN. Low-light high-resolution imaging will also be used to correlate cytoskeletal dynamics with force application and traction stresses on the substrate. In the second aim, the roles of proteins involved in the cadherin-dependent mechanosensitive regulation of cell polarity and protrusive activity will be investigated using loss-of-function (e.g., antisense morpholinos) and biochemical approaches. The initial focus of these experiments will be proteins involved in linking cadherins to the intermediate filament cytoskeleton (e.g., plakoglobin, desmoplakin and other cell-cell junctional components). Collective cell migration is critical to the progression of a variety of normal physiological and pathological states that include embryonic development, wound healing, cancer and metastasis, inflammation, and a wide range of congenital birth defects. This proposal will seek to broaden our understanding of the molecular mechanisms involved in this important mode of cellular migration.